Voltage-dependent potassium channels (Kv) are integral membrane proteins that, in response to membrane voltage changes, catalyze potassium ions to diffuse across the cell membrane. Kv channels regulate membrane excitability and are essential to many physiological processes, such as the rhythmic beating of heart, the communication between neurons, and the secretion of hormones. In a cell, Kv channels are always assembled with other proteins, and channel function is regulated by cellular environment through these associated proteins. The beta subunit (Kvbeta), a potential aldo-keto reductase (AKR), attaches to the intracellular side of the Kv1 family channels. They are ubiquitous in the brain and abundantly expressed in heart, and have been implicated in control of cell excitability and in sensing cellular redox state. The long-term goal of this research is to understand the role of Kvbeta in cell biology. We have recently identified two Kvbeta substrates and for the first time demonstrated that Kvbeta is a functional AKR. We also found that the substrates, when perfused to the intracellular side of the channel, modulate channel functions only when Kvbeta is co-expressed. These exciting new results led us to hypothesize that Kv1 channel functions are coupled to Kvbeta enzymatic activities. In this proposal we focus on screening for Kvbeta substrates and we propose the following two specific aims: Specific Aim 1. To identify small-molecule substrates of Kvbeta by a high throughput screen (the primary assay). We will use recombinant purified Kvbeta protein to screen for substrates in an enzymatic assay that follows the consumption of NADPH. Specific Aim 2. To test in an electrophysiological assay the positive hits from the screen (the secondary assay). We will test positive hits from Aim 1 using an electrophysiological assay that monitors channel functional change. Small-molecule substrates of Kvbeta will be used as a tool in our combined approaches of biochemistry, Xray crystallography, and electrophysiology to dissect molecular details of the coupling mechanism. The substrates will also provide a reasonable starting point for developing a new class K channel modulators. [unreadable] [unreadable]